Isothiocyanate and thiourea derivatives of benzoyl ecgonine conjugated to polypeptides

ABSTRACT

Novel benzoyl ecgonine derivatives are provided having an isothiocyanate group for conjugation to polypeptides and proteins to provide derivatives as reagents in immunoassays or for preparation of reagents for immunoassays. Particularly, the isothiocyanate is conjugated to enzymes or antigenic polypeptides or proteins. The antigenic polypeptides or proteins are employed for preparing antibodies to benzoyl ecgonine or cocaine, while the conjugated enzymes are used in immunoassays, where the enzyme is the detector molecule. The enzyme conjugate is found to have a high degree of sensitivity for the detection of benzoyl ecgonine, a cocaine metabolite.

United States Patent [191 Soffer et al.

[ Nov. 4, 1975 IS OTHIOCYANATE AND THIOUREA DERIVATIVES OF BENZOYL ECGONINE CONJUGATED T0 POLYPEPIIDES Inventors: Michael J. Softer, Redwood City;

Richard S. Schneider, Sunnyvale, both of Calif.

Assignee: Syva Corporation, Palo Alto, Calif.

Filed: June 13, 1973 Appl. No.: 369,658

[1.8. Cl 260/121; 23/230 B; 195/63; 195/68; 195/103.5; 260/78 A; 260/112 R; 260/112 B; 260/292; 260/293.54; 424/12;

Int. Cl. C07g 7/00; C07g 7/02; C08h l/OO Field of Search... 260/112 R, 121, 292, 293.54,

References Cited UNITED STATES PATENTS 9/1972 Goldstein et al 23/230 Primary ExaminerHoward E. Schain [57] ABSTRACT Novel benzoyl ecgonine derivatives are provided having an isothiocyanate group for conjugation to polypeptides and proteins to provide derivatives as reagents in immunoassays or for preparation of reagents for immunoassays. Particularly, the isothiocyanate is conjugated to enzymes or antigenic polypeptides or proteins. The antigenic polypeptides or proteins are employed for preparing antibodies to benzoyl ecgonine or cocaine, while the conjugated enzymes are used in immunoassays, where the enzyme is the detector molecule. The enzyme conjugate is found to have a high degree of sensitivity for the detection of henzoyl ecgonine, a cocaine metabolite.

5 Claims, No Drawings ISOTHIOCYANATE AND THIOUREA DERIVATIVES OF BENZOYL ECGONINE CONJUGATED TO POLYPEPTIDES BACKGROUND OF THE INVENTION 1. Field of the Invention The use of cocaine for other than medicinal uses has become of increasing concern. Cocaines availability has increased with concomitant efforts to police its use and prevent its distribution from unauthorized sources. In addition, in clinics providing drug therapy, it is important that the therapist be aware whether the person being treated has terminated or is continuing the use of drugs.

There has been continuing efforts to find reliable and rapid ways for detecting the use of drugs. It is also nec' essary that any method be able to distinguish cocaine or its metabolite from other drugs. Where urine is tested, it is found that cocaine is metabolized to benzoyl ecgonine. Therefore, in order to detect the use of cocaine, it is necessary to detect the presence of benzoyl ecgonine.

2. Description of the Prior Art A number of immunoassay systems suggest themselves as useful for the detection of cocaine or its metabolites. Radioimmunoassay has been reported as useful in the detection of opiate alkaloids. See U.S. Pat. N0. 3,709,868. U.S. Pat. No. 3,690,834 discloses a system using a free radical detector for immunoassays. U.S. Pat. application Ser. No. 143,609, filed May 14, 1971, now abandoned discloses an immunoassay technique employing enzymes, sold under the trademark EMIT, by Syva Corporation. Odell, Competitive Protein Binding, Blackwell Scientific Publications, Oxford, 1971, discloses a number of conjugated haptens to proteins, see particularly Chapter 2, beginning at page 25. A wide variety of functionalities are disclosed as useful for conjugation.

SUMMARY OF THE INVENTION DESCRIPTION OF THE SPECIFIC EMBODIMENTS The compounds of this invention are thiocarbamoyl derivatives of benzoyl ecgonine or its methyl ester, as well as the antibodies prepared from the antigenic conjugates of the benzoyl ecgonine derivative with an antigenic poly(amino acid).

For the most part, the compounds of this invention will have the following formula:

n is the number of benzoyl ecgonine groups conjugated to the poly(amino acid) and on the average will be at least 1 and not :more than the molecular weight of B divided by 500, more usually not more than the molecular weight of B divided by 2000,

and usually not exceeding 250. The thiocarbamoyl group may be meta or para, usu ally para.

When a and B are taken together, the compound will have the following formula:

CO R l yes it. wherein:

R is hydrogen or methyl, usually hydrogen, and the isothiocyanate group is either meta or para, normally para.

The isothiocyanate compound is prepared by esterifying the appropriate ecgonine derivative with the appropriate nitrobenzoic acid. The ester may then be reduced to the aminobenzoate, conveniently employing catalytic hydrogenation. The amino compound may then be derivatized to the isothiocyanate using thiophosgene. The amino compound is combined with the thiophosgene under mild conditions.

Conjugation of the isothiocyanate to a poly(amino acid) is carried out by combining the appropriate ratio of the isothiocyanate to the poly(amino acid) under mild conditions and at a constant mildly basic pH, normally in the range of about 8 to 9.

Of particular interest for the use of the subject compound is the conjugation to an amino group, which is part of a poly(amino acid) structure. By poly(amino acid) is intended both polypeptides and proteins. One group of poly(amino acids) is antigenic, so that by conjugation of the isothiocyanate to the antigenic poly(amino acid) a product is obtained which can be used in the formation of antibodies to benzoyl ecgonine. A narrow class of poly(amino acids), which can also be used as antigens, though not normally be used as such, are enzymes which are employed as the detector in an immunoassay system.

Polypeptides usually encompass from about 2 to 100 amino acid units (usually less than about 12,000 molecular weight). Larger polypeptides are arbitrarily called proteins. Proteins are usually composed of from 1 to 20 polypeptide chains, called subunits, which are associated by covalent or non-covalent bonds. Subunits are normally of from 100 to 300 amino acid groups (approximately 10,000 to 35,000 molecular weight). For the purposes of this invention, polypeptide is intended to include individual polypeptide units or polypeptides which are subunits of proteins, whether composed solely of polypeptide units or polypeptide units in combination with other functional groups, such as porphyrins, as in hemoglobin or cytochrome oxidase.

The first group of poly(amino acid) materials which will be considered are the antigenic poly(amino acids). These may be joined to the isothiocyanate group through an amino group. The thiourea product, which may include some thiourethane, can be used for the formation of antibodies to cocaine metabolites. The poly(amino acid) materials which may be used vary widely, normally being from about 1,000 to million molecular weight, more usually from 12,000 to 1 million molecular weight, and most frequently from about 50,000 to 500,000 molecular weight.

With most conventional poly(amino acids), there will not be more than about one benzoyl ecgonine or derivative group per 1,500 molecular weight of poly(amino acid), usually not more than one group per 2,000 molecular weight. There will be at least one group per 500,000 molecular weight, usually at least one per 50,000 molecular weight. With intermediate molecular weight antigens (50,000 to 1 million), the number of benzoyl ecgonine or derivative groups will generally be from about 2 to 250, usually from 10 to 100.

With low molecular weight antigens, 1,000 to 5,000 molecular weight, the number of benzoyl ecgonine or derivative groups will be in the range of l to 10, usually in the range of 2 to 5. Therefore, there may be as many as one benzoyl ecgonine or derivative group per 500 molecular weight of poly(amino acid).

Usually, the number of groups bonded to the poly(amino acid) will be related to the available amino groups, e.g., the number of lysines present. While the benzoyl ecgonine or derivative group may be bonded through the isothiocyanate to hydroxyl or mercaptan groups, which are present in the polypeptide, for the most part the bonding will be to amino and, therefore, the compounds are described as thioureas. However, thionoesters may also be present.

Amino acids present in poly(.amino acids) which have free amino groups for bonding to the isothiocyanate modified benzoyl ecgonine or derivative thereof, include lysine, arginine, histidine, etc. The hydroxylated and mercpatan substituted amino acids include serine, cystene and threonine.

Various protein types may be employed as the antigenic material. These types include albumin, serum proteins, e.g., globulins, ocular lens proteins, lipoproteins, etc. Illustrative proteins include bovine 'y-globulin, etc. Small natural polypeptides which are immunogenic, such as gramicidin may also be employed. Various synthetic polypeptides may also be employed, such as polymers of lysine, glutamic acid, phenylalanine, tyrosine, etc., either by themselves or in combination. Of particular interest is polylysine or a combination of lysine and glutamic acid. Any synthetic polypeptide must contain a sufficient number of active groups, as for example, amino groups provided by lysine.

The second group of poly(amino acids) are the enzymes to which the isothiocyanate derivative may be conjugated. As indicated, the benzoyl ecgonine derivative modified enzyme is useful for immunoassays. The immunoassay technique will follow in more detail.

Various enzymes may be used such as oxidoreductases, hydrolases, lyases, and the like. These enzymes include esterases, amidases, phosphorylases, carbohydrases, oxidases, reductases and the like. Of particular interest are such enzymes as lysozyme, amylase, dehydrogenases, particularly malate dehydrogenase, lactate dehydrogenase, mannitol-l-phosphate dehydrogenase, and glucose 6-phosphate dehydrogenasee, B-glucuronidase, cellulase, and phospho-lipas e, particularly phospholipase C. The enzymes will usually have molecular weights in the range of about 1 X 10 to 6 X 10 more usually in the range of about 1.2 X 10 to 3 X 10 There will usually be at least one benzoyl ecgonine or derivative group per enzyme molecule and usually not more than one group per 1,500 molecular weight, usually not more than one group for 2,000 molecular weight. Usually, there will be at least one benzoyl ecgonine or derivative group per 50,000 molecular weight, and more usually at least one group per 30,000 molecular weight. The modified enzyme will retain on the average at least 10%, more usually at least 30% of the original activity of the unmodified enzyme.

Where the benzoyl ecgonine or derivative is bonded to a poly(amino acid), there need be only one benzoyl ecgonine or derivative group, but usually there will be at least two groups. With the enzymes, the number of benzoyl ecgonine or derivative groups will generally be of from I to 30, more usually 2 to 25. Usually, there will be at least two, more usually at least three groups per enzyme, when the enzyme is randomly substituted with the benzoyl ecgonine or derivative groups, and preferably not more than 16 groups.

The substituted poly(amino acid)s will, for the most part, have the following formula:

member and from 0 to l of the heteroatoms, i.e., oxygen and nitrogen, as annular members.

The molecules bonded to the isothiocyanate will normally be of from 8 to 16 carbon atoms, usually of from 8 to 12 carbon atoms. The functionality for linking to the ecgonine derivative may be bonded directly to an annular carbon atom or bonded to an annular carbon atom through an aliphatic chain of from 1 to 4 carbon atoms, usually of from 1 to 2 carbon atoms. The molecules may have from O to 2 sites of ethylenic unsaturation, more usually from 0 to 1 site of ethylenic unsaturation.

For the most part, the stable nitroxide functionalities which are employed will have the following formula:

wherein:

X is a divalent aliphatic radical, having from 0 to l site'of aliphatic unsaturation, usually aliphatically saturated of from 1 to 6 carbon atoms, usually from 2 to 3 carbon atoms being annular atoms;

A is lower alkyl (1 to 6, usually one to 3 carbon atoms), preferably methyl; and

Y is of the following formula:

ANTIBODIES The preparation of antibodies specific for haptenic materials is a well-established practice. A thorough description of the procedure may be found in Williams et al, Methods in Immunology and lmmunochemistry, Ac ademic Press, New York and London, 1967, pages 197 to 385, particularly that portion beginning at page 197 and ending at page 254. I

For preparation of antibodies to haptens, a hapten is conjugated to an antigenic material such as a polypeptide or protein, although polysaccharides, particularly containing aminosugars, can also be used.

The particular manner in which the hapten is bonded to the antigenic material will depend on the functionalities which are available on the haptenic material and the antigenic material, the number of haptenic groups to be conjugated to the antigenic material, and the like. Groups which find use include carboxy groups, which may be activated by employing the mixed carbonic acid anhydride or carbodiimide, imidates, diazo groups, ahaloketones, and the like. Numerous procedures for the conjugation of a wide variety of haptens have been developed and published.

The antigenic conjugate may be injected in the fluid state; adsorbed to insoluble particles, such as alumina; or incorporated in matrix materials such as agar, calcium alginate, or Freunds adljuvants (complete or incomplete, depending on whether mycobacteria are incorporated). The adsorbtion to various insoluble colloidal carriers is described in the aforementioned text, the carriers being illustrated by alumina, aluminum phosphate, blood charcoal and the like. Other materials include polyacrylamide gel, bentonite, and protein. As adjuvants, methylated bovine serum albumin and Freunds adjuvant find use. Complete Freunds adjuvant is a water-in-oil emulsion, using emulsion stabilizers such as lanolin, lanolin derivatives, e.g., Aquaphor, mannide mono-oleate and Arlacel A, available from Duke Laboratories, South Newark, Connecticut. The complete adjuvant is distinguished from the incomplete adjuvant by having mycobacteria, e.g., M.butyricum or M.tuberculo sis. The adjuvants are commercially available from Difco Laboratories, Detroit, Michigan.

Immunization can be carried out in a variety of ways with a number of different animals. For the most part, for commercial production of antibodies, relatively large animals are employed, such as equine, bovine, porcine, canine, ovine, caprine, rodentia, rabbits and hares. Of particular interest are horses, goats, sheep and cows, that is, the larger domestic animals, as well as rabbits.

The antigenic material mayv be injected interperitoneally, intramuscularly, subcutaneously, and the like. When employing Freunds adjuvants, usually in combination with saline, the amount of antigen employed will vary depending on the particular antigenic material and the number and period of prior injections. Usually, about 0.1 to 5 mg of antigenic material will be employed per one ml of solution. The total amount of antigenic material and solution will depend on the size, nature and weight of the animal employed. The initial injection will normally be at a number of sites, aliquots of the composition being employed.

The first injections of antigen serve to load the animal, and a period of time is allowed to pass before booster injections are introduced, normally 2 to 8 weeks. Bleeding may occur after each injection, so as to follow the formation of the desired antibody. Depending on the animal, bleedings can be carried out via heart puncture, the carotid artery or external jugular 7 with calcium chloride, with clotting resulting. 1f necessary, thrombin may be added to enhance clotting. After breaking up the clot, the clot is compressed and serum is withdrawn and filtered. Various other procedures are known and can be employed. 1

The serum can be treated in various ways, depending on its subsequent use. The serum may be fractionated by employing ethanol, Rivanol, neutral salts, such as ammonium sulfate or sodium sulfate, or the like. Usually, the product will be dialyzed after dissolution in a buffer, filtered and then isolated. Alternatively, the serum may be chromatographed on various modified cellulose columns, e.g., diethylaminoethylcellulose or carboxymethylcellulose. Various physical means may be employed to concentrate the desired antibodies.

Numerous preservatives can be employed to stabilize the antibodies and the antibodies will normally be stored at reduced temperatures.

The antibodies are primarily 'y-globulin which are found to have a molecular weight of about 150,000. The antibodies will be specific for a particular spatial structure and polar-non-polar distribution. Varying structures deviating from an ideal structure will give different binding constants.

EXPERIMENTAL The following examples are offered by way of illustration and not by way of limitation.

(All temperatures that are not otherwise indicated are in Centigrade).

EXAMPLE 1 Preparation of para-Aminobenzoylecgonine A. Ecgonine hydrochloride (5.5g, 24.8 mmoles) was dissolved in 35ml of methanol (dried over 3-A Molecular sieves) and saturated with dry hydrogen chloride keeping the receiver cool by immersion in an ice bath. Upon saturation the receiver was heated to 40 for 0.5 hour and evaporated to dryness in vacuo. The white residue was stored at 0.05mm Hg over potassium hydroxide for 16 hours and then dissolved in the minimum amount of hot methanol to which 200ml of boiling acetone was quickly added. After cooling in ice and filtering, there was obtained 4.2g of white crystals, m.p. 214-2l5 (lit. 2l42l5). Evaporation of the motherliquor and repetition of the recrystallization yielded 0.8g m.p. 2l2-214. Total yield was 86.3% of theory.

B To 20ml of cold saturated potassium carbonate solution in a 125ml separator funnel was added a solution of 5.0g (213 mmoles) ecgonine methyl ester hydrochloride in ml water. The aqueous mixture was extracted with 4 X 60ml of chloroform. The combined chloroform extracts were dried over anhydrous sodium carbonate and evaporated in vacuo. Pumping at 0.05mm Hg for min. yielded 4.0g (93%) of TLC pure (:1 CHCl :MeOH) ecgonine methyl ester.

The 4.0g (20:1 mmoles) ecgonine methyl ester was dissolved in 50ml dry benzene and then ml benzene was distilled off. To the cooled distillation pot was added 3.65ml triethylamine and a solution of 3.72g freshly recrystallized p-nitrobenzoyl chloride in 5ml of dry benzene was added dropwise with cooling (ice bath) and agitation.

The resulting sludge was stirred at 40 for 1 hr under nitrogen. After cooling to room temperature the reaction mixture was taken up in 100ml of chloroform and washed with 3 X 20ml 5% aqueous sodium carbonate 8 solution. The chloroform solution was dried over sodium carbonate, evaporated in vacuo and pumped (0.05mmHg) on overnight to yield 5.7g (85.3%) of yellow oil [one spot on TLC"(/5, CHCl /MeOH)] with same R, as known sample but having a slight odor of triethylamine. No further attempt at purification was made, and the product was used directly in the next step.

C. To a solution of 6.5g p-nitrococaine in 250ml absolute methanol was added 600mg 10% Pd/C under an N blanket. The resulting mixture was hydrogenated at atmospheric pressure with rapid stirring and slight heating from the magnetic stirrer. After 0.5 hr. H up-' take ceased, [1.530 liters, calculated is 1.440 liters without correction for atmospheric pressure]. The catalyst was removed by suction filtration over a Celite pad in a fritted glass funnel (medium grade). The resulting clear solution was evaporated in vacuo to approximately 75m] and heated to dissolve crystals which formed and then allowed to cool to room temperature, followed by cooling in ice and filtering to give 4.0g white crystals, m.p. 188189. The mother-liquor was concentrated to 3ml, cooled in ice and filtered. After washing the crystals with 6ml of cold methanol, there was obtained 1.2g powdery crystals, m.p. 185l88. Total yield 88%.

D. p-Aminococaine (2.08g) in 15ml of water was refluxed with rapid stirring under nitrogen for 6 hours. The solution was allowed to cool to room temperature and then cooled in ice and filtered. The crystals were washed with 5ml cold water and dried at 0.05mm Hg for 2 hours to yield 1.2g clear needlelike crystals, m.p. 287 (dec.). The compound slowly turns brown upon exposure to air and light. Recrystallization of 200mg from 2ml boiling water gave an analytically pure sample.

Calc, C, 63.14; H, 6.62; N, 920.

Found, C, 63.32; H, 6.62; N, 9.16.

EXAMPLE 11 Preparation of para-isothiocyanato Benzoyl Ecgonine lnto two ml of 2N hCl under nitrogen was introduced mg (0.33 mmole) of para-aminobenzoyl ecgonine. To the solution was added 31 1.1 (46mg) of thiophosgene and the heterogeneous mixture stirred vigorously under nitrogen at room temperature. After 10 minutes, the thiophosgene could no longer be observed. The product crystallized. The mixture was cooled in ice, filtered, and the filtrate washed with water. After drying the solid over phosphorous pentoxide and potassium hydroxide, 62mg was isolated. m.p. 257 (dec).

To the mother-liquor was added approximately 3ml of water, the solution cooled in ice and the precipitate collected. The second crop yielded 66mg. m.p. 257.

Anal. Calcd for C H N O S Cl: C, 53.33;

H, 5.02; N, 7.32; S, 8.36; Cl, 9.27. Fd:

C, 53.39; H, 5.14; N, 7.32; S, 8.41; Cl, 9.01.

EXAMPLE III Conjugation of para-lsothiocyanatobenzoyl Ecgonine to Lysozyme A solution of 60mg (25p. mole) lysozyme in 5.0m! waterwas cooled to 4C and adjusted to pH 9.0 with 0.05M NaOH. A total of 99.5mg (25y. mole) p-isothiocyanatobenzoyl ecgonine was added in one portion to the alkaline protein solution. This conjugation was run on the pH-STAT at 4 with the machine maintaining the pH at 9.0 with 0.05M NaOH. (The reaction can also be performed by the manual addition of base using a pH meter to follow the course of the reaction). After 3% hours, the clear solution was adjusted to pH 9.5. Since no precipitation occurred, the pH was lowered to 7.0 with dilute HCl. The clear solution was dialyzed against water for 48 hours. The dialysate was immediately suitable for the assay of benzoyl ecgonine.

EXAMPLE 1V Conjugation of para-lsothiocyanatobenzoyl Ecgonine to Glucose-6-Phosphate Dehydrogenase (G-6-PDH) To a 0.5ml solution of G-6-PDH (2mg protein/ml) in 0.05M phosphate buffer at pH 7.0 at 4 was added dropwise a solution of 1.4 mg para-isothiocyanatobenz' oyl ecgonine in 0.5ml of0.05M tris at pH 7.0. The reaction mixture was stirred at 4 at pH 7.0 for 30 minutes before the pH was adjusted to 8.6 by the addition of 0.05M sodium hydroxide. After 5 hours, the mixture was dialyzed against 2 liters 0.055M tris pH 7.9.

The resulting dialysate was assayed for enzyme activity in the presence of NAD and glucose-6-phosphate by measuring the increase in absorption at 340nm. That benzoyl ecgonine had been attached to the enzyme was shown by the change in enzyme activity in the presence and absence of benzoyl ecgonine antibodies.

Enzyme Rate Amount of Benzoyl Ecgonine The addition of 50p.l of antibody results in a 34% inhibition of enzyme activity.

The procedure for the G-6-PDH assay is as follows:

The following reagents are employed: 0.1M NAD in H O, pH adjusted to 5-6 with 0.05M NaOl-l; 0.066M glucose-6-phosphate (G-6-P) in 0.055M .Tris-Cl, pH 7.9; 0.055M Tris-Cl buffer, pH 7.9 with 0.1% rabbit serum albumin.

Into a spectrophotometer cell is metered 1.1 NAD, 5011.1 G-6-P, specified amount of antibody solution, 10111 of a 1:4 dilution of the enzyme dialysate and sufficient buffer to bring the total volume to lml. The optical density is read at 340nm for 5 minutes and the enzyme rate determined by averaging the rate between the second and fifth minute. The temperature is maintained at 37.

To demonstrate the use of the subject compounds, immunoassays were carried out.

Assays The immunoassay employing an enzyme as the detector is carried out as follows. With lysozyme, a bacterial suspension of M.luteus is employed, dissolving 0.2m] of a suspension of 300mg of the bacteria in 400ml of 0.025M, pH6,Tris-maleate buffer. First the bacterial suspension is introduced into the assay vessel. When testing a sample, 50p] of the sample is then introduced. This is followed by 5011.1 of antibody solution in 0.025M, pl-I6, Tris-maleate buffer and the transfer made quantitative by washing with 325 11 of the same buffer solution. The benzoyl ecgonine conjugate to lysozyme (50 .tl) is then added to give a binding site to 10 benzoyl ecgonine ratio of about 121.5 and 325 1.1 of buffer used to insure quantative transfer.

The results are then read by observing the decrease in optical density at 436nm for 40 seconds at 36. The results are reported in OD/mirt. In the subject assay, the antibody employed was obtained in response to a conjugate of para-diazabenzoyl ecgonine with bovine serum albumin. The binding constant was about 1 X 10 M. The concentration of antibody was 6.9 X 10' m, based on binding sites as determined employing a free radical assay technique with a cocaine spin label.

Following the procedure described above, a number of drugs were studied for cross reactivity in the benzoyl ecgonine assay. The following table indicates the results. The results are reported as the amount of cross reacting drug necessary to produce a response equivalent to 1.0 pg per ml benzoyl ecgonine.

Methamphetamine Employing the benzoyl ecgonine conjugate to lysozyme in accordance with the previously described method, the assay was found to have excellent sensitivity and reproducibility of results in the range of 0.5 to 5 pg per ml concentration of benzoyl ecgonine. Ten urine samples were each spiked to levels of 1 pg per ml and 5 .Lg per ml of benzoyl ecgonine. Each sample was assayed in triplicate and the average value of each sample was used to calculate the experimental concentration and the percent recovery. The recovery observed contained some contribution of spiking error, operator error, and the urine variation. The average recovery of activity lies around The following tables indicate the results.

Samples Spiked to 1.0 ng/ml Benzoyl Ecgonine Samples Spiked to 5.0 #g/ml Benzoyl Ecgonine Average Sample OD Units ug/ml Recovery -continued Samples Spiked to 5.0 pg/ml Benzoyl Ecgohine Average Sample OD Units #g/ml '7? Recovery Mean 151 6.01 pg/ml 120% Recovery 128-179 15.6-10.5 #g/ml 72-210% Range Samples Not 2 and No. 5 give an extraordinarily high recovery of benzoyl ecgonine. Since the blanks for these two samples are negligible and also since this magnitude of urine variation is beyond normal limits. some error in the spiking of these two samples must have occurred.

The lysozyme benzoyl ecgonine conjugate employed in the subject assay is found to be stable for long periods of time when stored at 4C. The enzyme retains a substantial proportion of the original activity after conjugation, so as to provide a high degree of activity in the assay. In addition, the enzyme conjugate is able to provide a substantial rate change when the amount of benzoyl ecgonine is varied from 0.5 to 5.0 ug/ml. Therefore, the subject conjugate provides an accurate, effective and rapid method for determining extremely small quantities of benzoyl ecgonine. Furthermore, the assay only requires 50ul or less of urine sample, so that extremely small amounts of benzoyl ecgonine are required for detection.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious 12 that certain changes and modifications may be practiced within the scope of the appended claims.

What is claimed is: 1. A conjugated antigenic poly(amino acid):

b/CHsi COR wherein:

R is hydrogen or methyl; PP is an antigenic poly(amino acid) of at least 1000 molecular weight bonded through amino groups to form a thiourea; and n is the number of groups bonded to the poly(amino acid), and in the range of about one to the molecular weight of the poly(amino acid) divided by 1000. 2. A conjugated poly(amino acid) according to claim 1, wherein R is hydrogen, n is in the range of 1 to 200, and PP has a molecular weight in the range of 12,000 to 1 million.

3. A conjugated poly( amino acid) according to claim 2, wherein PP is bovine serum albumin.

4. An antibody prepared in response to a conjugate according to claim 1.

5. An antibody prepared in response to a conjugate according to claim 3. 

1. A CONJUGATED ANTIGENIC POLY(AMINO ACID):
 2. A conjugated poly(amino acid) according to claim 1, wherein R is hydrogen, n'' is in the range of 1 to 200, and PP has a molecular weight in the range of 12,000 to 1 million.
 3. A conjugated poly(amino acid) according to claim 2, wherein PP is bovine serum albumin.
 4. An antibody prepared in response to a conjugate according to claim
 1. 5. An antibody prepared in response to a conjugate according to claim
 3. 